The standard uptake value (SUV), commonly used to quantify F-18-FluoroDeoxyGlucose (FDG) uptake in small animal brain PET imaging, is affected by many factors. In this study the influence of fasting times, inter-scan duration and repetitive scanning on the variability of different Shy measures is investigated. Additionally it is demonstrated that these variables could adversely influence the outcome of a pharmacological challenge when not accounted for. Naive Sprague-Dawley rats (n = 20) were randomly divided into five different fasting groups (no fasting up to 24 h of fasting). SUV brain uptake values were reproducible in naive animals when a fasting period of at least 12 h is used and for shorter fasting periods SUV values need to be corrected for the glucose level. Additionally, a separate animal group (n = 6) was sufficiently fasted for 16 h and in a longitudinal setting being scanned six times in three weeks. Especially with short inter-scan durations, increasing glucose levels were found over time which was attributed to increased stress due to repeated food deprivation, altered food intake or scan manipulations. As a result, even with controlled and sufficient fasting, blood glucose levels should be taken into account for data quantification. Strikingly, even the brain activation effects of an NMDA-antagonist challenge with memantine could not be detected in experiments with a short inter-scan duration if glucose levels were not taken into account. Correcting for glucose levels decreases the inter- and intra-animal variability for rat brain imaging. SUV corrected for glucose levels yields the lowest inter-animal variation. However, if the body weight changes significantly, as in a long experiment, quantification based on the glucose corrected percentage injected dose (and not SUV) is recommendable as this yields the lowest intra-animal variation. (C) 2014 Elsevier Inc. All rights reserved.